Mammals express four Notch receptor paralogs (Notchi -4) that have been implicated in a broad range of functions during embryogenesis, post-natal development, and vartous pathologies, including cancer. As a result, there is growing interest in therapies aimed at blocking or activating Notch signaling in specific cellular or pathological contexts. During the previous funding period, we explored the spectrum of effects triggered by loss of Notchi activity with a novel genetic approach combining a knock-in Notchi allele (Nl ::CreLo), in which Cre replaced the NICD, and a conditional Notchi target allele (Niflox). The trans-heterozygous animals (Nl :CreLo/flox) were bred to reporter mice developed in the Molecular Reporter Core which contained floxed-stop click beetle red luciferase and dual reporter (lac-Z or eYFP) genes on respective alleles downstream ofthe ROSA26 locus. A systematic, life-long in vivo screen with bioluminescence imaging of animals wherein a random, progressive and age-dependent Notchi loss occurred identified a role for Notchi in suppressing neoplasia ofthe vascular system. Our findings caution against the Genetech approach of therapies targeting Notchi and provide a model in which to assess strategies to delay, prevent or control vascular tumor formation and thereby widen the therapeutic window of anti-Notch therapies. We will examine age, diet and organ dependence in vascular tumor formation using new Nl ::CreERT2 alleles permitting temporal control over loss of Notchi. We will use another new Notch trans-heterozygous combination, N2::CreLo/flox, to monitor risks associated specifically with loss of Notch2. An additional benefit from the proposed experiments will be a detailed map of all stem cell compartments where Notchi and Notch2 maintain stemness or promote differentiation. These studies will ultimately lead to safer Notch- targeting therapies.